The present invention relates to polynucleotide assays, especially for diagnostic purposes, and to kits and polynucleotide reagent complexes for such assays.
U.S. patent application Ser. No. 607,885 of S. E. Diamond et al, filed May 7, 1984 and assigned jointly to Allied Corporation and Genetics Institute, Inc., describes a polynucleotide displacement assay for target nucleotide sequences of sample nucleic acids. In such assays, a probe polynucleotide contains a segment (the target binding region) complementary to the target nucleotide sequence to be determined. A second polynucleotide, called the Labeled Polynucleotide or the Signal Strand, is bound by complementary base pairing to at least a portion of the target binding region. In use, such a reagent complex containing probe polynucleotide and signal strand is contacted by a sample; target nucleotide sequences in the sample bind to the target binding region of the probe polynucleotide and displace the signal strand from the reagent complex. Displaced signal strands are then detected, generally after a separation step which involves in many cases a probe polynucleotide which is either immobilized to a solid support before the sample is brought in or is immobilized to a solid support after the displacement step. Only limited embodiments are disclosed which can be conducted in a homogenous manner (without a separation step).
Such displacement assays have various advantages (as described in U.S. application Ser. No. 607,885) over prior hybridization assays represented by U.S. Pat. No. 4,358,535 to Fllkow et al (1982) in that difficulties associated with immobilizing the sample nucleic acid are eliminated. For most readouts (determinations of the displaced labeled polynucleotide or signal strand), however, a separation step is required.
Application U.S. Ser. No. 729,503 of C. Vary et al, filed May 2, 1985, describes a heterogenous assay of the displacement polynucleotide type wherein the displaced signal strand (labeled polynucleotide) has a digestable polyribonucleotide segment, especially at its 3' end. After displacement by target nucleotide strand and separation, such signal strands are digested (especially by the enzyme polynucleotide phosphorylase) to ribonucleoside phosphates (especially diphosphates) and the adenosine phosphates so produced (especially adenosine diphosphate) are determined. Such determination proceeds especially by phosphorylation to adenosine triphosphate (ATP) and determination of the ATP (e.g., with the luciferase catalyzed reaction with luciferin) or of the by-product of the phosphorylation step (e.g., pyruvate determination using NADH and lactate dehydrogenase). See also U.S. application Ser. No. 729,502 of C. Vary et al, filed May 2, 1985, for a further discussion of the digestion, phosphorylation and determination steps which may be used to assay displaced signal strands containing a digestable polyribonucleotide segment.